In gene therapy and artificial substance production systems using living organisms, means of introducing a nucleic acid, a protein, and such, into the interior of a cell are extremely important. On the other hand, techniques for extracting structures such as the nucleus of a cell, are also gaining wide attention. Therefore, it can be said the injecting and extracting substances into/from cells, the basic unit constituting organisms, is a fundamental technique of bioengineering.
In the prior art, the method of destroying or perforating the membrane depends on a physical shearing force. However, cell membrane destroying using the shear force of a capillary requires skilled experience on the part of manipulator. Also, in many cases, the capillary cannot be inserted into the cell membrane of normal cells, except large size egg cells, due to the flexibility of the cell membrane. In this regard, as a method that does not depend on the physical shear force, a method of denaturing a membrane with a substance (a membrane-denaturing agent) inducing a membrane-denaturing reaction is presented (PCT/JP99/01223). The membrane-denaturing agent is a substance that induces the membrane-denaturing reaction by a selected specific stimulus as a trigger. By controlling a degree of membrane denaturation, a perforating method allowing the self-repairing of the perforated membrane can be provided.
According to the method, the membrane-denaturing agent must be in contact with the cell or close thereto. In one embodiment, a photosensitizer as a membrane-denaturing agent is in contact with a site of cell via a capillary, and light is applied to the cell-neighboring area as a whole (for example, a circular region of about 100 micrometers including the cell contacting the capillary).
In this method, the light exposure is introduced such that an area including the capillary that contacts the membrane-denaturing agent is exposed. Therefore, if the capillary is filled with a mixture of a substance to be injected into the cell and the membrane-denaturing agent, the membrane-denaturing agent within the capillary as well as the membrane-denaturing agent on the tip of the capillary may be activated by the light application, thereby generating reactive oxygen species.
If the substance filled in the capillary to be injected into the cell is a chemically vulnerable substance such as genes and such, the substance may be damaged by the reactive oxygen species.
To avoid such damage, a light application method that allows a pinpoint light application to the tip of capillary may be employed. However, the method has disadvantages in that a light focus adjusting mechanism that follows the movement of the tip of capillary should be required and it makes the apparatus costly.
Therefore, the present invention relates to an improvement of the membrane-denaturing method with a membrane-denaturing substance inducing the membrane-denaturing reaction. An object of the present invention is to eliminate the influence of membrane-denaturing agent to the substance to be injected into the cell as low as possible.
Objects of the present invention are not limited to the above-mentioned object. For example, when the capillary or intracellular sensor is used in a tissue in vivo and such, it is difficult to apply stimulus such as the light to the tip of the capillary or sensor locally. Another object of the present invention is to provide a method and apparatus that enable locally applying the stimulus, with a simple construction.